Enhancers of CFTR chloride channel function

ABSTRACT

Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) is essential for opening the CFTR chloride channel. A short segment containing many negatively charged amino acids (817-838, NEG2) within the regulatory (R) domain of CFTR is a critical regulator of the chloride channel activity. Deletion of NEG2 from CFTR completely eliminates the PKA dependence of the chloride channel. Exogenous NEG2 peptide interacts with the CFTR molecule and exhibits stimulatory effects on CFTR function. Our data suggest that NEG2 interacts with other cytosolic domains of CFTR to control the opening transitions of the chloride channel.

[0001] This invention was made with government support under RO1 HL/DK49003, P30 DK27651 and RO1 DK51770 awarded by the National Institute ofHealth. The government has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention is related to the field of cystic fibrosis. Moreparticularly, it is related to the area of therapeutic treatments anddrug discovery for treating cystic fibrosis.

BACKGROUND OF THE INVENTION

[0003] Defects in CFTR, a chloride channel located in the apicalmembrane of epithelial cells, are associated with the common geneticdisease, cystic fibrosis (Quinton, 1986, Welsh and Smith, 1993,Zielenski and Tsui, 1995). CFTR is a 1480 amino acid protein that is amember of the ATP binding cassette (ABC) transporter family (Riordan etal., 1989, Higgins, 1992). Each half of CFTR contains a transmembranedomain and a nucleotide binding fold (NBF), and the two halves areconnected by a regulatory, or R domain. The R domain is unique to CFTRand contains several consensus PKA phosphorylation sites (Cheng et al.,1991, Picciotto et al., 1992).

[0004] Opening of the CFTR channel is controlled by PKA phosphorylationof serine residues in the R domain (Tabcharani et al., 1991, Bear etal., 1992) and ATP binding and hydrolysis at the NBFs (Anderson et al.,1991, Gunderson and Kopito, 1995). Phosphorylation adds negative chargesto the R domain, and introduces global conformational changes reflectedby the reduction in the α-helical content of the R domain protein(Dulhanty and Riordan, 1994). Thus, electrostatic and/or allostericchanges mediated by phosphorylation are likely to be responsible forinteractions between the R domain and other CFTR domains that regulatechannel function (Rich et al., 1993, Gadsby and Naim, 1994).

[0005] Rich et al., 1991 showed that deletion of amino acids 708-835from the R domain (ΔR-CFTR), which removes most of the PKA consensussites, renders the CFTR channel PKA independent, but the openprobability of ΔR-CFTR is one-third that of the wild type channel anddoes not increase upon PKA phosphorylation (Ma et al., 1997, Winter andWelsh, 1997). Thus, it is possible that deletion of the R domain removesboth inhibitory and stimulatory effects conferred by the R domain onCFTR chloride channel function. This conclusion is supported by studiesthat show that addition of exogenous unphosphorylated R domain protein(amino acids 588-858) to wt-CFTR blocks the chloride channel (Ma et al.,1996), suggesting that the unphosphorylated R domain is inhibitory.Conversely, exogenous phosphorylated R domain protein (amino acids588-855 or 645-834) stimulated the ΔR-CFTR channel, suggesting that thephosphorylated R domain is stimulatory (Ma et al., 1997, Winter andWelsh, 1997). Therefore, it appears that the manifest activity(stimulatory or inhibitory) depends on the phosphorylation state of theR domain.

[0006] About 25% of the known 700 mutations in CFTR produce a mutantCFTR protein which is properly transported to the apical membrane ofepithelial cells but have only low level, residual channel activity.There is a need in the art for agents which can boost the level ofchannel activity in those mutants having low level activity.

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to provide an isolatedpolypeptide useful for enhancing the open probability of CFTR chloridechannels.

[0008] It is another object of the present invention to provide a methodof activating a CFTR protein to enhance its open probability.

[0009] These and other objects of the invention are achieved byproviding one or more of the embodiments described below. In oneembodiment of the invention an isolated polypeptide is provided. Thepolypeptide comprises a portion of CFTR (cystic fibrosis transmembraneconductance regulator) protein of between 10 and 100 amino acids, saidportion comprising 18 amino acids as shown in SEQ ID NO: 1.

[0010] In another embodiment of the invention a method is provided foractivating a CFTR protein. A polypeptide is applied to a CFTR proteinwhich forms a cAMP regulated chloride channel. The polypeptide consistsof a portion of CFTR protein which comprises 18 amino acids as shown inSEQ ID NO: 1, whereby the open probability of the channel formed by theCFTR increases by at least 25%.

[0011] According to another aspect of the invention a method is providedfor activating a CFTR protein. A polypeptide is applied to a CFTRprotein which forms a cAMP regulated chloride channel. The polypeptideconsists of a portion of CFTR protein which comprises 22 amino acids asshown in SEQ ID NO: 2, whereby the open probability of the channelformed by the CFTR increases by at least 25%.

[0012] The present invention thus provides the art with reagents andtools for enhancing function of channels which are defective in cysticfibrosis patients.

DETAILED DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1. Deletion of Negatively Charged Regions from the R DomainResults in Expression of Mature Glycosylated, Phosphorylatable CFTRProteins

[0014] (FIG. 1A) Sequences of NEG1 and NEG2 within the R domain.Residues where, mutations have been identified in the CFTR cDNA areunderlined (E822K, E826K, D836Y).

[0015] (FIG. 1B) NEG2 is predicted to form an amphipathic α-helix asdetermined by secondary structure determination (Geourion and Deleage,1995, Rost and Sander, 1993, Rost and Sander, 1994) and illustrated inthis space filling model. Negatively charged residues are colored pink,and the positively charged lysine is colored green.

[0016] (FIG. 1C) In vitro phosphorylation of wt—(lane 1), ΔNEG1—(lane 2)and ΔNEG2-CFTR (lane 3) by PKA in the presence of γ-³²P-ATP. Both thecore (band B) and fully glycosylated (band C) forms of all three CFTRmolecules are phosphorylated.

[0017]FIG. 2. ΔNEG2-CFTR Forms a Chloride Channel that is Unregulated byPKA

[0018] (FIG. 2A) Single channel currents of wt, ΔNEG1- and ΔNEG2-CFTRincorporated into the lipid bilayer. While activities of wt- andΔNEG1-CFTR absolutely require the presence of PKA in thecis-intracellular solution, the ΔNEG2-CFTR channel opens without PKAphosphorylation.

[0019] (FIG. 2B) Diary plot of ΔNEG2-CFTR channel open probabilityversus time shows that addition of 200 units/ml of PKA, a maximallystimulating concentration, does not affect channel activity. The dashedline indicates the average open probability for each segment of theexperiment. Channels were recorded at −100 mV.

[0020]FIG. 3. The Synthetic NEG2 Peptide both Stimulates and InhibitsCFTR

[0021] (FIG. 3A) Diary plot (open probability versus time) of a wt-CFTRchannel illustrating the effect of the NEG2 peptide on the openprobability of the channel in the planar lipid bilayer. Theconcentration of synthetic NEG2 in the cis-intracellular solution isindicated above the plot.

[0022] (FIG. 3B) Single channel currents from the wt-CFTR channel wereacquired at −80 mV at the points indicated in A. The cis-intracellularsolution contained 2 mM ATP and 50 units PKA/ml.

[0023] (FIG. 3C) Single channel trace from ΔNEG2-CFTR incorporated intothe lipid bilayer membrane. Traces were acquired at −80 mV. Thecis-solution contained 2 mM ATP and no PKA. The top two traces wereacquired before synthetic NEG2 peptide addition, with the second tracebeing an expansion of the first. In the bottom two traces, 0.44 μM ofthe NEG2 peptide has been added and stimulation is observed. The closedtime visibly decreases after peptide addition.

[0024]FIG. 4. NEG2 Enhances CFTR Channel Activity by Increasing theOpening Rate of the Channel

[0025] Histograms of open and closed events of the wt-CFTR channel at−80 mV were generated without peptide (control, left panel) and with 4.4μM NEG2 peptide in the cis-solution (right panel).

[0026] (FIG. 4A) The open time histograms contain a single exponentialcomponent with a time constant of 124 ms (control) and 105 ms(peptide-stimulated).

[0027] (FIG. 4B) The closed time histograms contain a fast component andmultiple slow components.

[0028] (FIG. 4C) The closed-burst duration histograms were constructedusing a delimiter of 40 ms (represented by the arrow in B). The solidlines in C represent the fit according to the double exponentialequation y=P₂* exp [t−α−exp αt−α)]₂+P₃* exp [t−α₃−exp(t−α₃)] whereα₂=log τ_(σ2)α₃=log τ_(τ3)P₂=probability of the intermediate closedcomponent, and P₃=probability of the long closed component. The best fitparameters are P₂=0.811, τ_(c2)=459 ms, P₃=0.189, τ_(c3)=2494 ms(control); P₂=0.957, τ_(c2)=105 ms, P₃=0.043, τ_(c3)=1652 ms(peptide-stimulated).

DETAILED DESCRIPTION OF THE INVENTION

[0029] It is a discovery of the present inventors that negativelycharged amino acids at the carboxyl terminal of the R domain (817-838,NEG2) is involved in both the stimulatory and inhibitory functions ofthe R domain on the chloride channel. Moreover, a polypeptide whichcontains this portion of the CFTR amino acid sequence can be used toenhance the open probability of both wild-type and minimally activemutant CFTR protein.

[0030] The isolated polypeptide according to the invention consists of aportion of CFTR (cystic fibrosis transmembrane conductance regulator)protein. The portion preferably contains at least 18 amino acids asshown in SEQ ID NO: 1. However, fewer amino acid residues of thesequence may be used if they retain the channel enhancing functiondescribed herein for the 18 and 22 residue polypeptides. See also SEQ IDNO: 2. Thus the polypeptide may be from about 10 or 15 amino acidresidues up to about 30 or even 100 amino acid residues. An isolatedpolypeptide may be synthetic or made in a recombinant organism. It maybe a proteolytic cleavage product of a larger primary expressionproduct, including full-length, wild-type CFTR. Preferably thepolypeptide will be free of full-length CFTR. The polypeptide willpreferably be free of other proteins and polypeptides as well. However,it may be desirable that the polypeptide be fused to another polypeptideto provide additional functional properties. For example, fusion toanother protein such as keyhole limpet hemocyanin would be used toincrease immunogenicity. Another desirable fusion partner is amembrane-penetrating peptide. Such peptides include VP-22 (SEQ ID NO:3), as well as the peptides shown in SEQ ID NO: 4 and SEQ ID NO: 5. Suchpeptides can be used to facilitate the uptake by target cells of thepolypeptide.

[0031] The polypeptides of the present invention can be used to enhancethe function of wild-type or minimally active mutant CFTR proteins. Thepolypeptide functions to decrease the closed time of the channels formedby CFTR. A polypeptide can be applied to the CFTR protein in anycontext. It can be applied in vitro or in vivo. If in vitro it can be toCFTR in cultured cells or to planar bilayer membranes containing CFTR.If in vivo, the polypeptide can be applied directly to airway epithelialcells. Such application can be by any means known in the art, includingbut not limited to using a gargle or a nebulizer to deliver aerosolizedpolypeptide to the target cells. In addition, the peptide can bedelivered in an indirect mode, by delivering a gene construct to theairway epithelial cells, which when taken up by the cells causes them toexpress the polypeptide. The delivery of the polypeptide to the CFTRpreferably increases the open probability of the channel formed by theCFTR by at least 25%. More preferably it increases the open probabilityby at least 50%, at least 75%, at least 100%, at least 125%, at least150%, or at least 200%.

[0032] A CFTR construct comprises a nucleic acid sequence encoding theamino acid sequence shown in SEQ ID NO: 1. A suitable promoter forexpression in lung epithelia is also desirable. Many such promoters areknown in the art, and any can be used as appropriate for a particularapplication.

[0033] It is believed that the administration of the polypeptide of thepresent invention will be the most useful in treatment of a class ofmutants which produce CFTR proteins which are properly delivered to theplasma membrane but which are only residually or minimally active. Knownmutants of CFTR are listed athttp://www.genet.sickkids.on.ca/cftr-cgi-bin/fulltable. One candetermine that a particular CFTR mutant is fully processed and reachesthe plasma membrane in a Western blot assay using antibody against CFTR.Fully processed mutants achieve mature glycosylation status and appearon the gel as “band C and band B” whereas mutants that are retained inthe endoplasmic reticulum are not fully glycosylated and show only “bandB”. See Example 2, below and FIG. 1C.

[0034] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLES Example 1

[0035] Deletion of a Negatively Charged Region (a.a. 817-838) From the RDomain of CFTR Alters PKA-Dependent Regulation of the CFTR Channel.

[0036] CFTR contains a large intracellular regulatory (R) domain wheremultiple PKA phosphorylation sites are located. There are two regionswithin the R domain that contain a high proportion of negatively chargedamino acids, a.a. 725-733 (NEG1) and a.a. 817-838 (NEG2). It is possiblethat these two regions could have allosteric or electrostaticinteractions with other regions of CFTR and thus affect its chloridechannel function. To test the role of NEG1 and NEG2, two deletionmutants, NEG1 -CFTR and NEG2-CFTR, were created. The CFTR mutants weretransiently expressed in HEK 293 cells, and their single channelfunctions were studied using the bilayer reconstitution system. Westernblots indicate that both NEG1-CFTR and NEG2-CFTR process normally andtraffic to the plasma membrane of HEK 293 cells. Both mutants formfunctional chloride channels in the bilayer membrane, with singlechannel conductances similar to the wt-CFTR channel. Like wt-CFTR,opening of NEG1-CFTR requires absolutely PKA phosphorylation and ATPbinding/hydrolysis. In contrast to wt-CFTR, opening of NEG2-CFTR doesnot require PKA phosphorylation. Thus, deletion of NEG2, but not NEG1,alters PKA-dependent regulation of the CFTR chloride channel. Our datasuggest that NEG2 could form a ‘putative gating particle’ of the CFTRchannel possibly through electrostatic and/or allosteric interactionswith other domains of CFTR.

Example 2

[0037] ΔNEG1- and ΔNEG2-CFTR are Glycosylated.

[0038] The R domain of CFTR contains two negatively charged regions,amino acids 725-733 (NEG1) and amino acids 817-838 (NEG2), that residein close proximity to two PKA phosphorylation sites, S737 and S813, usedin vivo (FIG. 1A) (Cheng, et al. 1991). NEG2 is predicted to form anamphipathic (-helical structure with a negatively charged face (FIG. 1B)(Geoudjon and Deleage, 1995, Rost and Sander, 1993, Rost and Sander,1994). Three mutations (E822K, E826K, D836Y), two of which were clearlyobtained from patients with CF (E822K and D836Y). have been identifiedwithin the NEG2 region that result in the removal of negative charges(www.genet.sickkids.on.ca). The E822K CFTR channel has a low openprobability relative to wt-CFTR (wild type-CFTR), but the E826K CFTRchannel has single channel properties similar to wt-CFTR (Vankeerberghenet al., 1998). The presence of these disease-causing mutations suggeststhe potential importance of the NEG2 region. To investigate the roles ofNEG1 and NEG2 in CFTR function, these regions were deleted from CFTRusing mutagenesis and subcloning. The ΔNEG1- and ΔNEG2-CFTR proteinswere transiently expressed in human embryonic kidney 293 cells. Membranevesicles containing the CFTR proteins were isolated and subjected toSDS-PAGE. Like wt-CFTR, both ΔNEG1- and ΔNEG2-CFTR are present both inthe core glycosylated (band B) and the fully glycosylated form (band C)(FIG. 1C).

Example 3

[0039] The Open Probability of the ΔNEG2-CFTR Chloride Channel is MuchLess Than That of Wild Type but is Independent PKA, Although it Containsall PKA Phosphorylation Sites.

[0040] Single channel measurements indicate that the ΔNEG1-CFTR channelis similar to wt-CFTR in its PKA dependence. No chloride channels areobserved in the absence of PKA (FIG. 2A) and the open probability in thepresence of PKA and ATP is similar to wt-CFTR. In contrast, theΔNEG2-CFTR channel opens without PKA (FIG. 2A). The constitutiveactivity of the ΔNEG2-CFTR channel is unlikely to be due to theendogenous phosphorylation of the ΔNEG2-CFTR protein, since proteinphosphatase 2A, which decreases activity of the wt-CFTR opened by PKAand ATP (Ma et al., 1997), has no effect on the ΔNEG2-CFTR channel(n=4). Moreover, addition of PKA up to 200 units/ml, four times theconcentration required to fully activate wt-CFTR (Ma et al., 1997), doesnot increase the open probability of the channel (FIG. 2B). ΔNEG2-CFTRhas conductance properties similar to wild type (Tao et al., 1996).However, the open probability of the ΔNEG2-CFTR chloride channel is muchless than that of wild type and cannot be increased by PKA (P_(o)=0.035(0.012 and P_(o)=0.026 (0.013 without and with PKA respectively, n=5).While NEG2 may represent an inhibitory region, removal of these aminoacids does not result in a fully activated channel. The failure of theΔNEG2-CFTR channel to respond to PKA does not result from inability ofthe channel to be phosphorylated, for an in vitro assay using (-³²P-ATPshowed comparable phosphorylation of wt-CFTR and ΔNEG2-CFTR (FIG. 1C).Thus, it appears that removal of NEG2 from CFTR completely eliminatesthe PKA dependence of the chloride channel, although the ΔNEG2-CFTRchannel still contains all ten PKA sites and can be phosphorylated.

Example 4

[0041] NEG2 Polypeptide Stimulates Both Wild-Type and a NEG2 CFTRProteins at Concentrations Greater than 0.44 μM.

[0042] To test whether the NEG2 region is responsible for bothstimulatory and inhibitory interactions between the R domain and otherdomains, synthetic NEG2, a 22 amino acid peptide, was added to thecis-intracellular side of single CFTR channels captured in the planarlipid bilayer (FIG. 3). The diary plot of open probability as a functionof time shows the activity of a single wt-CFTR channel during the courseof the experiment (FIG. 3A). After peptide addition, there are periodsof intense stimulation that last 4 to 8 minutes. These stimulatoryperiods are followed by either a return to the basal level of activitybefore peptide addition, or by an almost complete inhibition of thechannel, where only a flickery 3 pS conductance is observed. Duringstimulation, the open probability more than doubles and more transitionsare observed between the open and closed states (FIG. 3B). Thestimulatory response was observed in 6 of 7 experiments atconcentrations ≧0.44 μM (the remaining channel was inhibited uponpeptide addition (4.4 μM) and no stimulation was seen). Profoundinhibition was observed in three channels at concentrations ≧4.4 μM.When the NEG2 peptide was added to the intracellular side of theΔNEG2-CFTR channel, which lacks its own endogenous NEG2 sequence, asimilar stimulatory response was observed (FIG. 3C).

Example 5

[0043] The NEG2 Peptide Decreases the Closed Time of the Wild-Type CFTRProtein.

[0044] In order to understand the mechanism responsible for the increasein open probability, the gating kinetics of wt-CFTR without peptide andduring stimulation by synthetic NEG2 were analyzed. The open timedistributions of the wt-CFTR did not change during peptide stimulation,as both control (without NEG2 peptide) and peptide-stimulated channelshad an open lifetime of approximately 120 ms (FIG. 4A). Thus, theincrease in the open probability is not due to a change in the closingrate of the channel. However, the closed time distribution for thestimulated channel is clearly shifted to the left compared to thecontrol channel (FIG. 4B). There are three components to the closedstate, a fast (τ_(c1)), an intermediate (τ_(c2)), and a long (τ_(c3))closed component. The fast closed component is probably due to closingswithin a burst (Carson et al., 1995). Therefore, to identify better theclosed times between bursts, a delimiter of τ_(c)=40 ms was set at thenadir between the fast and intermediate closed times (illustrated by thearrow in FIG. 4B) to generate the closed-burst duration histograms. Asshown in FIG. 4C, following peptide stimulation, the intermediate closedtime was reduced from 459 ms to 105 ms, whereas the long closed timeremained relatively unchanged. Thus, the interaction of NEG2 with CFTRincreased the intermediate-opening rate of the channel. This increase inopening rate is similar to that observed when the phosphorylated Rdomain protein (amino acids 645-834) was added to CFTR-ΔR/S660A inexcised inside-out patches (Winter and Welsh, 1997). Additionally,modification of C832, which resides within NEG2, by N-ethylmaleimide(NEM) results in irreversible stimulation of PKA-phosphorylated CFTRchloride channel activity (Cotten and Welsh, 1997), further emphasizingthe importance of NEG2 in CFTR regulation.

[0045] These data, taken together, show that the NEG2 region confersboth stimulatory and inhibitory functions of the R domain on the CFTRchannel. When this region is deleted from CFTR, the resultant channelopens without PKA (loss of inhibitory function), but it never achievesopen probability comparable to wild type even when phosphorylated withPKA (loss of stimulatory function). This same sequence, expressed as apeptide, results in stimulation of channel openings at lowerconcentrations and profound inhibition of channel activity at higherconcentrations, when added to the intracellular side of CFTR channels.It seems likely that this sequence interacts with CFTR at differentsites on the nucleotide binding domains to either stimulate or inhibitchannel openings. Phosphorylation of the R domain, in this model,changes its conformation and thus presents the NEG2 sequence better tothe stimulatory than the inhibitory site. A current model for channelopening is that phosphorylated channels open in response to ATP bindingand hydrolysis at the first nucleotide binding fold (NBF1) (Gadsby andNairn, 1994, Ma and Davis, 1998). Since stimulation by NEG2 occurs byincreasing channel openings, a likely site of stimulation is NBF1,though other models are possible.

METHODS USED IN EXAMPLES 1-5

[0046] Subcloning of CFTR Gene

[0047] The wt, ΔNEG1-, and ΔNEG2-CFTR cDNAs were subcloned into anEpstein-Barr virus-based episomal eukaryotic expression vector, pCEP4(Invitrogen, San Diego, Calif.), between the Nhel and Xhol restrictionsites. The ΔNEG1 and ΔNEG2 deletion mutants were created using thepALTER mutagenesis system and shuttled from pALTER into pCEP4 bysubstituting the corresponding fragment in pCEP4 wt-CFTR with the mutantfragment between the Xhol and BstZ171 restriction sites. The ΔNEG1-CFTRcDNA has 27 bases deleted (amino acids 725-733). The ΔNEG2-CFTR cDNA has66 bases deleted (amino acids 817-838).

[0048] Expression of CFTR in HEK 293 Cells

[0049] A human embryonic kidney cell line (293-EBNA HEK; Invitrogen) wasused for transfection and expression of the CFTR proteins (Ma et al.,1997, Ma et al., 1996, Xie et al., 1995). The HEK-293 cell line containsa pCMV-EBNA vector, which constitutively expresses the Epstein-Barrvirus nuclear antigen-1 (EBNA-1) gene product and increases thetransfection efficiency of Epstein-Barr virus-based vectors. The cellswere maintained in Dulbecco's Modified Eagle Medium with 10% FBS and 1%L-glutamine. Geneticin (G418, 250 (g/ml) was added to the cell culturemedium to maintain selection of the cells containing the PCMV-EBNAvector. Lipofectamine reagent (Life Technologies, Inc) in Optimem media(serum-free) was used to transfect the HEK-293 cells with pCEP4(wt),pCEP4(ΔNEG1), or pCEP4(ΔNEG2). After 5 hours, serum was added to themedia (10% final serum concentration). Twenty-four hours aftertransfection, the transfection media was replaced with fresh media. Thecells were harvested two days after transfection and microsomal membranevesicles were prepared for single channel measurements in the lipidbilayer reconstitution system.

[0050] Vesicle Preparation From Transfected HEK 293 Cells

[0051] HEK-293 cells transfected with pCEP4(CFTR) were harvested andhomogenized using a combination of hypotonic lysis and Douncehomogenization in the presence of protease inhibitors (Ma et al., 1997,Ma et al., 1996, Xie et al., 1995). Microsomes were collected bycentrifugation of postnuclear supernatant (4500×g, 15 min) at 100,000×gfor 20 min and resuspended in a buffer containing 250 mM sucrose, 10 mMHEPES, pH 7.2. The membrane vesicles were stored at −75° C. until use.

[0052] In Vitro Phosphorylation of CFTR Proteins

[0053] CFTR proteins isolated in membrane vesicles were bound to proteinG agarose using a mouse monoclonal anti-human CFTR antibody (Genzyme).The protein G agarose was washed, and (-³²P-ATP (10 (Ci) and proteinkinase A (˜10 units/50(l) was added. Samples were incubated at 30(C. forone hour during phosphorylation. Excess (γ-³²P-ATP was removed, andSDS-PAGE sample buffer (200 mM Tris-Cl, pH 6.7,9% SDS, 6%beta-mercaptoethanol, 15% glycerol, and 0.01% bromophenol blue) wasadded to denature CFTR and release it from the protein G agarose. Thesamples were subjected to electrophoresis on a 5% SDS-polyacrylamidegel, transferred to a polyvinylidene difluoride membrane, and exposed tofilm.

[0054] Preparation of NEG2 Peptides

[0055] The 22 amino acid peptide corresponding to NEG2 was custom madeby Quality Controlled Biochemicals. Inc. The peptide was resuspended inwater to a concentration of 1 mg/ml and pH was adjusted to aphysiological range (7.2-7.4) using KOH and HCl. The space filling modelof the NEG2 peptide was generated, based on secondary structurepredictions (Geou jon and Deleage, 1995, Rost and Sander, 1993, Rost andSander, 1994), using the Insight II program from Molecular SimulationsIncorporated.

[0056] Reconstitution of CFTR Channels in Lipid Bilayer Membranes

[0057] Lipid bilayer membranes were formed across an aperture of ˜200 (mdiameter with a mixture ofphosphatidylethanolamine:phosphatidylserine:cholesterol in a ratio of5:5:1. The lipids were dissolved in decane at a concentration of 33mg/ml. The recording solutions contained: cis (intracellular), 200 mMCsCl, 1 mM MgCl₂, 2 mM ATP, and 10 mM HEPES-Tris (pH 7.4); trans(extracellular), 50 mM CsCl, 10 mM HEPES-Tris (pH 7.4). Vesicles (1-4(l) containing either wild-type, ΔNEG1-, or ΔNEG2-CFTR were added to thecis solution. The PKA catalytic subunit was present at a concentrationof 50 units/ml in the cis solution unless noted otherwise. Singlechannel currents were recorded with an Axopatch 200A patch clamp unit(Axon Instruments). The currents were sampled at 1-2.5 ms/point. Singlechannel data analyses were performed with pClamp and TIPS softwares.

[0058] References

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1 5 1 18 PRT Homo sapiens 1 Gly Leu Glu Ile Ser Glu Glu Ile Asn Glu GluAsp Leu Lys Glu Cys 1 5 10 15 Phe Phe 2 22 PRT Homo sapiens 2 Gly LeuGlu Ile Ser Glu Glu Ile Asn Glu Glu Asp Leu Lys Glu Cys 1 5 10 15 PhePhe Asp Asp Met Glu 20 3 559 PRT HSV-1 3 Met Ala Arg Phe His Arg Pro SerGlu Asp Glu Asp Asp Tyr Glu Tyr 1 5 10 15 Ser Asp Leu Trp Val Arg GluAsn Ser Leu Tyr Asp Tyr Glu Ser Gly 20 25 30 Ser Asp Asp His Val Tyr GluGlu Leu Arg Ala Ala Thr Ser Gly Pro 35 40 45 Glu Pro Ser Gly Arg Arg AlaSer Val Arg Ala Cys Ala Ser Ala Ala 50 55 60 Ala Val Gln Pro Ala Ala ArgGly Arg Asp Arg Ala Ala Ala Ala Gly 65 70 75 80 Thr Thr Val Ala Ala ProAla Ala Ala Pro Ala Arg Arg Ser Ser Ser 85 90 95 Arg Ala Ser Ser Arg ProPro Arg Ala Ala Ala Asp Pro Pro Val Leu 100 105 110 Arg Pro Ala Thr ArgGly Ser Ser Gly Gly Ala Gly Ala Val Ala Val 115 120 125 Gly Pro Pro ArgPro Arg Ala Pro Pro Gly Ala Asn Ala Val Ala Ser 130 135 140 Gly Arg ProLeu Ala Phe Ser Ala Ala Pro Lys Thr Pro Lys Ala Pro 145 150 155 160 TrpCys Gly Pro Thr His Ala Tyr Asn Arg Thr Ile Phe Cys Glu Ala 165 170 175Val Ala Leu Val Ala Ala Glu Tyr Ala Arg Gln Ala Ala Ala Ser Val 180 185190 Trp Asp Ser Asp Pro Pro Lys Ser Asn Glu Arg Leu Asp Arg Met Leu 195200 205 Lys Ser Ala Ala Ile Arg Ile Leu Val Cys Glu Gly Ser Gly Leu Leu210 215 220 Ala Ala Ala Asn Asp Ile Leu Ala Ala Arg Ala Gln Arg Pro AlaAla 225 230 235 240 Arg Gly Ser Thr Ser Gly Gly Glu Ser Arg Leu Arg GlyGlu Arg Ala 245 250 255 Arg Pro Met Thr Ser Arg Arg Ser Val Lys Ser GlyPro Arg Glu Val 260 265 270 Pro Arg Asp Glu Tyr Glu Asp Leu Tyr Tyr ThrPro Ser Ser Gly Met 275 280 285 Ala Ser Pro Asp Ser Pro Pro Asp Thr SerArg Arg Gly Ala Leu Gln 290 295 300 Thr Arg Ser Arg Gln Arg Gly Glu ValArg Phe Val Gln Tyr Asp Glu 305 310 315 320 Ser Asp Tyr Ala Leu Tyr GlyGly Ser Ser Ser Glu Asp Asp Glu His 325 330 335 Pro Glu Val Pro Arg ThrArg Arg Pro Val Ser Gly Ala Val Leu Ser 340 345 350 Gly Pro Gly Pro AlaArg Ala Pro Pro Pro Pro Ala Gly Ser Gly Gly 355 360 365 Ala Gly Arg ThrPro Thr Thr Ala Pro Arg Ala Pro Arg Thr Gln Arg 370 375 380 Val Ala ThrLys Ala Pro Ala Ala Pro Ala Ala Glu Thr Thr Arg Gly 385 390 395 400 ArgLys Ser Ala Gln Pro Glu Ser Ala Ala Leu Pro Asp Ala Pro Ala 405 410 415Ser Thr Ala Pro Thr Arg Ser Lys Thr Pro Ala Gln Gly Leu Ala Arg 420 425430 Lys Leu His Phe Ser Thr Ala Pro Pro Asn Pro Asp Ala Pro Trp Thr 435440 445 Pro Arg Val Ala Gly Phe Asn Lys Arg Val Phe Cys Ala Ala Val Gly450 455 460 Arg Leu Ala Ala Met His Ala Arg Met Ala Ala Val Gln Leu TrpAsp 465 470 475 480 Met Ser Arg Pro Arg Thr Asp Glu Asp Leu Asn Glu LeuLeu Gly Ile 485 490 495 Thr Thr Ile Arg Val Thr Val Cys Glu Gly Lys AsnLeu Leu Gln Arg 500 505 510 Ala Asn Glu Leu Val Asn Pro Asp Val Val GlnAsp Val Asp Ala Ala 515 520 525 Thr Ala Thr Arg Gly Arg Ser Ala Ala SerArg Pro Thr Glu Arg Pro 530 535 540 Arg Ala Pro Ala Arg Ser Ala Ser ArgPro Arg Arg Pro Val Glu 545 550 555 4 27 PRT Artificial Sequencemembrane permeating peptide 4 Gly Trp Thr Leu Asn Ser Ala Gly Tyr LeuLeu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala Leu Ala Lys LysIle Leu 20 25 5 16 PRT Artificial Sequence membrane permeating peptide 5Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 1015

1. An isolated polypeptide comprising a portion of CFTR (cystic fibrosistransmembrane conductance regulator) protein of between 10 and 100 aminoacids, said portion comprising 18 amino acids as shown in SEQ ID NO: 1.2. The polypeptide of claim 1 which comprises 22 amino acids as shown inSEQ ID NO:
 2. 3. The polypeptide of claim 1 wherein the polypeptide isfused to a membrane-penetrating peptide.
 4. The polypeptide of claim 2wherein the polypeptide is fused to a membrane-penetrating peptide. 5.The polypeptide of claim 3 wherein the membrane-penetrating peptide isselected from the group consisting of: VP-22 (SEQ ID NO: 3), (SEQ ID NO:4), and (SEQ ID NO: 5).
 6. The polypeptide of claim 4 wherein themembrane-penetrating peptide is selected from the group consisting of:VP-22 (SEQ ID NO: 3), (SEQ ID NO: 4), and (SEQ ID NO: 5).
 7. Thepolypeptide of claim 1 which is free of phosphorylation.
 8. A method ofactivating a CFTR protein comprising: applying a polypeptide to a CFTRprotein which forms a cAMP regulated chloride channel, said polypeptidecomprising a portion of CFTR protein of between about 10 and 100 aminoacids, said portion comprising 18 amino acids as shown in SEQ ID NO: 1,whereby the open probability of the channel formed by the CFTR increasesby at least 25%.
 9. The method of claim 8 wherein the open probabilityof the channel formed by the CFTR increases by at least 50%.
 10. Themethod of claim 8 wherein the open probability of the channel formed bythe CFTR increases by at least 75%.
 11. The method of claim 8 whereinthe open probability of the channel formed by the CFTR increases by atleast 100%.
 12. The method of claim 8 wherein the open probability ofthe channel formed by the CFTR increases by at least 125%.
 13. Themethod of claim 8 wherein the open probability of the channel formed bythe CFTR increases by at least 150%.
 14. The method of claim 8 whereinthe open probability of the channel formed by the CFTR increases by atleast 200%.
 15. The method of claim 8 wherein the CFTR protein is amutant which reaches a cell's plasma membrane but fails to undergo fullactivation.
 16. The method of claim 15 wherein the CFTR protein islisted at http://www.genet.sickkids.on.ca/cftr-cgi-bin/fulltable. 17.The method of claim 8 wherein the step of applying is performed byadministering an aerosolized polypeptide to a patient with a mutant CFTRprotein.
 18. The method of claim 8 wherein the CFTR protein is in alipid bilayer and a change in conductance is measured upon applying thepolypeptide.
 19. The method of claim 8 wherein the step of applying thepolypeptide is accomplished by administering a nucleic acid encoding thepolypeptide to a patient who expresses the CFTR protein, whereby thepolypeptide is expressed
 20. The method of claim 19 wherein the nucleicacid is administered as an aerosol to the patient's airways.
 21. Amethod of activating a CFTR protein comprising: applying a polypeptideto a CFTR protein which forms a cAMP regulated chloride channel, saidpolypeptide comprising a portion of CFTR protein of between 10 and 100amino acids, said portion comprising 22 amino acids as shown in SEQ IDNO: 1, whereby the open probability of the channel formed by the CFTRincreases by at least 25%.
 22. The method of claim 21 wherein the openprobability of the channel formed by the CFTR increases by at least 50%.23. The method of claim 21 wherein the open probability of the channelformed by the CFTR increases by at least 75%.
 24. The method of claim 21wherein the open probability of the channel formed by the CFTR increasesby at least 100%.
 25. The method of claim 21 wherein the openprobability of the channel formed by the CFTR increases by at least125%.
 26. The method of claim 21 wherein the open probability of thechannel formed by the CFTR increases by at least 150%.
 27. The method ofclaim 21 wherein the open probability of the channel formed by the CFTRincreases by at least 200%.
 28. The method of claim 21 wherein the CFTRprotein is a mutant which reaches a cell's plasma membrane but fails toundergo full activation.
 29. The method of claim 28 wherein the CFTRprotein is listed athttp://www.genet.sickkids.on.ca/cftr-cgi-bin/fulltable.
 30. The methodof claim 21 wherein the step of applying is performed by administeringan aerosolized polypeptide to a patient with a mutant CFTR protein. 31.The method of claim 21 wherein the CFTR protein is in a lipid bilayerand a change in conductance is measured upon applying the polypeptide.32. The method of claim 21 wherein the step of applying the polypeptideis accomplished by administering a nucleic acid encoding the polypeptideto a patient who expresses the CFTR protein, whereby the polypeptide isexpressed.
 33. The method of claim 32 wherein the nucleic acid isadministered as an aerosol to the patient's airways.
 34. The method ofclaim 8 or 21 wherein the polypeptide is free of phosphorylation.